CN116284332A

CN116284332A - Peptidoglycan recognition protein-D, preparation method and application thereof

Info

Publication number: CN116284332A
Application number: CN202310199196.0A
Authority: CN
Inventors: 张嵘; 王夏璐; 张景海
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2023-03-03
Filing date: 2023-03-03
Publication date: 2023-06-23

Abstract

The invention discloses a peptidoglycan recognition protein-D, a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention uses protein separation and purification technology to separate and purify natural peptidoglycan recognition protein-D from tussah, analyzes the primary structure (gene and protein), uses gene engineering technology to realize the expression of peptidoglycan recognition protein-D and its derivative or analogue or partial fragment gene in host cell, purifies to obtain recombinant peptidoglycan recognition protein-D and its derivative or analogue or partial fragment, and immunizes animals to obtain corresponding antibody. The natural and recombinant peptidoglycan recognition protein-D, derivatives or analogues or partial fragments thereof and antibodies thereof can be widely applied to the biomedical fields of prevention, detection diagnosis, treatment and the like of microorganisms and microorganism related molecular patterns.

Description

Peptidoglycan recognition protein-D, preparation method and application thereof

Technical field:

the invention belongs to the technical field of biological medicine, and relates to a structure of peptidoglycan recognition protein-D, an obtaining method thereof, a novel physiological function and application thereof in the field of biological medicine. In particular, the invention relates to the structure of peptidoglycan recognition protein-D and analogues, active fragments thereof, a preparation method and functions thereof, and application thereof in the biomedical fields of microorganism and related molecular pattern detection and diagnosis, promotion of insect hemolymph melanin generation, influence on insect synthesis of different types of antibacterial peptides and the like, and preparation of peptidoglycan recognition protein-D and analogues or active fragment antibodies thereof and application thereof in the biomedical fields.

The background technology is as follows:

the peptidoglycan recognition protein (Peptidoglycan recognition protein, PGRP) is a member of the family of pattern recognition receptors (pattern recognition receptor, PPR) and recognizes the pathogen-associated molecular pattern (Pathogen-related molecular patterns, PAMPs) existing only on the surface of pathogenic organisms to realize the perception of the foreign pathogens, and then selectively activates Toll pathway, IMD pathway, JAK-STAT pathway, active oxygen metabolism or blackening reaction and other pathways to remove the pathogens.

Peptidoglycan recognition proteins were first discovered in silkworms in 1996 by Yoshida et al (Yoshida, H.; kinoshita, K.; ashida, M.Purification of a Peptidoglycan Recognition Protein from Hemolymph of the Silkworm, bombyx Mori.J.biol. Chem.1996,271 (23), 13854-13860.) followed by PGRPs in succession in molluscs, echinoderms and vertebrate species. PGRPs contain one or more PGRP domains consisting of about 165 amino acid residues. This domain is homologous to N-acetylmuramic acid-alanine amidase and has about 30% similarity to phage T7 lysozyme with amidase activity. By structural analysis, it was found that: the PGRP domain is an inverted L-shaped groove structure consisting of five β -sheets in the central region and three α -helices in the periphery. PGRPs are classified into different types according to the molecular weight of the protein and amidase activity. PGRPs are classified into long, medium and short forms according to the molecular weight of the protein. PGRPs can be classified into catalytically active PGRPs and non-catalytically active PGRPs according to whether they have amidase activity or not. The PGRPs with catalytic activity can cut the amide bond between the N-acetylmuramic acid and the L-lysine of the peptidoglycan, crack the peptidoglycan into smaller fragments without immunostimulation activity, and further reduce the irritation of bacteria to IMD pathways. Whereas non-catalytically active PGRPs are unable to cleave peptidoglycans, but can be involved as recognition receptors in other immune reactions.

TmPGRP-D of yellow meal worm can bind to PGN and interact with TmGNBP1 and serine proteinase MSP to activate

Precursor shear enzyme, will->

Cleavage of the precursor to mature +.>

Binds to Toll receptors in the Toll pathway and activates the Toll pathway to synthesize antibacterial peptides. Similar to yellow meal worm, PGRP-D, SD and gram-negative binding protein-1 (GNBP-1) synergistically detect bacterial cell wall extracellular Lys-type PGN in drosophila, initiating activation of Toll pathway; whereas BmPGRP-S1 is involved in activating IMD pathways to induce AMPs expression. The phenol oxidase cascade results in melanin formation involved in the insect's defense against invading pathogenic microorganisms. Some PGRPs with receptor function can participate in the activation of the phenol oxidase cascade by recognizing microorganisms or PAMPs. The blackening reaction can be initiated by over-expression of DmPGRP-LE with receptor recognition function and continuous activation of DmPGRP-LC with receptor recognition function. In silkworms, bmPGRP-S1 can participate in the activation of phenol oxidase cascade reaction after being combined with PGN; in the tobacco astronomical moth, msPGRP-1 not only can recognize PGN, but also can participate in the phenol oxidase cascade reaction of the tobacco astronomical moth; corn borer PGRP-S can activate a phenol oxidase cascade upon binding S.aureus and B.thuringiensis; cotton bollworm PGRP-A may also be involved in the activation of the phenol oxidizing enzyme cascade. Drosophila secreted PGRP-SC1, -SC2, and-LB have amidase activity and cleave PGN molecules. 3 PGRP genes, PGLYRP-2 (or zfPGRP 2), PGLYRP-5 (zfPGRP-SC) and PGLYRP-6 (zfPGRP 6), respectively, were identified in zebra fish, and all of the 3 PGRP genes exhibited amidase activity and antibacterial activity.

The peptidoglycan recognition protein is a very conservative pattern recognition protein from lower animals to higher animals, and has great significance in host immune regulation and immune related disease research. However, there is currently no study on the structure, preparation, biological function and application of the tenuiform-D of the peptidoglycan recognition protein of insects of the family Lepidoptera (Lepidoptera).

The invention comprises the following steps:

the invention relates to a preparation method, a primary structure (genes and proteins), biological functions and application thereof of natural PGRP-D aiming at PGRP-D in lepidoptera (large) silkworm moth insects, and recombinant PGRP-D, analogues or active fragments thereof and biological functions and application thereof are obtained by utilizing a genetic engineering technology. In addition, the use of natural, recombinant PGRP-D and its analogues or active fragments as antigens to stimulate the production of antibodies by the body has been investigated.

The invention firstly utilizes the protein extraction, separation and purification technology to separate and purify the natural PGRP-D from lepidoptera (large) silkworm moth insects. Next, the primary structure (gene and protein) of PGRP-D was analyzed and its gene was obtained by using protein chemistry techniques and molecular biology techniques. And thirdly, utilizing a genetic engineering technology to realize the expression of the PGRP-D gene in host cells, and combining protein extraction, separation and purification technologies to obtain the recombinant PGRP-D. Meanwhile, the analogue or partial fragment of PGRP-D is obtained by utilizing the gene recombination technology. The natural and recombinant PGRP-D and analogues or partial fragments thereof can specifically identify a plurality of microorganism related molecular modes such as lipopolysaccharide, beta-1, 3-glucan, peptidoglycan, lipoteichoic acid, mannans and the like and microorganisms such as bacteria, fungi and the like. The combination activates the pro-phenoloxidase activating system in the humoral immunity of the insects on one hand and affects the synthesis of different types of insect antibacterial peptides on the other hand. The natural and recombinant PGRP-D, analogues or partial fragments thereof and the biological functions of the antibodies thereof can be widely applied to the biological medicine fields of prevention, detection diagnosis, treatment and the like aiming at microorganisms; meanwhile, the natural and recombinant PGRP-D and the analogues or partial fragments thereof induce insects to express a large amount of antibacterial peptide, so that the prepared antibacterial peptide can be applied to the biological medicine fields such as prevention, detection diagnosis and treatment of microorganisms.

The insect of the invention is lepidoptera, preferably a Nepalidae (Saturn iidae) insect, selected from tussah, castor-bean silkworm, nepal silkworm, indian tussah, amber silkworm, america tussah, ailanthus altissima silkworm, dashan silkworm, nepal silkworm, laurada, maple silkworm, and the insect is natural or artificial stocking or artificial breeding insect in any region. In order to provide a more complete and clear understanding of the present invention to those skilled in the art, the following description is presented with tussah as representative, and the selection of tussah as representative is not intended to limit the scope of the claims of the present invention in any way.

The PGRP-D, PGRP-D active fragment is obtained by utilizing genetic engineering expression, and comprises (1) an expression vector of a prokaryotic system, wherein an expression host cell is an escherichia coli cell or a bacillus subtilis cell or a lactobacillus, (2) an expression vector of a yeast cell system, the expression host cell is a yeast cell, (3) an expression vector of an insect cell system, the expression host cell is an insect cell, and (4) an expression vector of a mammalian cell system, and the expression host cell is a mammalian cell. The expression form is expressed in a cell or a secretion form, and the expression product in the expression system is used as a source for preparing PGRP-D, PGRP-D analogues or active fragments.

The "host cell" includes both prokaryotic cells and eukaryotic cells, and examples of commonly used prokaryotic host cells include E.coli, bacillus subtilis, and the like. Common eukaryotic host cells include yeast cells, insect cells, mammalian cells, and the like.

The microorganisms and their related molecular patterns referred to in the present invention are fungi, gram-positive and gram-negative bacteria and their related molecular patterns. In order for those skilled in the art to more fully and clearly understand the present invention, the following will be described with pichia pastoris, candida albicans, staphylococcus aureus, escherichia coli, micrococcus luteus, bacillus subtilis, etc. as representatives of microorganisms (fungi, gram positive bacteria, and gram negative bacteria), lys-PGN, DAP-PGN, lipoteichoic acid, mannans, beta-1, 3-glucan, lipopolysaccharide, etc. as representatives of microorganism-related molecular patterns, and the selection of the above-described specific microorganisms or specific microorganism-related molecular patterns as representatives is not intended to limit the scope of the claims of the present invention in any way.

The invention is realized by the following technical scheme:

the invention provides a peptidoglycan recognition protein-D, the amino acid sequence of which is shown in SEQ ID NO: 1.

Based on the above technical scheme, further, the peptidoglycan recognition protein-D is derived from Lepidoptera (Lepidoptera) insects of the family sarcinidae (samurniidae) and is selected from one of tussah, castor, wild silkworm, indian tussah, amber, us tussah, ailanthus, wild silkworm, american wild silkworm, camphorwood silkworm and maple silkworm.

In another aspect, the invention provides a gene encoding said peptidoglycan recognition protein-D.

Based on the technical scheme, further, the nucleotide sequence of the peptidoglycan recognition protein-D gene is shown as SEQ ID NO: 2.

In another aspect, the present invention provides a derivative or analogue or active fragment of peptidoglycan recognition protein-D, comprising an amino acid sequence as set forth in SEQ ID NO:1 and has the biological activity of peptidoglycan recognition protein-D.

Based on the technical scheme, further, the derivative or analogue or active fragment of the peptidoglycan recognition protein-D is selected from Met-PGRP-D sequence, met-His ₆ tag-PGRP-D sequence, met-PGRP-D-His ₆ Tag sequence, met-His ₆ Tag-thrombin cleavage site-PGRP-D sequence, met-GST tag-thrombin cleavage site-PGRP-D sequence, met-PGRP-D-thrombin cleavage site-GST tag sequence, met-PGRP-D-Flag tag sequence, met-Flag tag-PGRP-D sequence, met-His ₆ tag-SUMO tag-thrombin cleavage site-PGRP-D sequence, met-SUMO tag-thrombin cleavage site-PGRP-D-His ₆ A tag sequence.

The invention also provides a preparation method of the peptidoglycan recognition protein-D, which uses one or more than two of haemolymph, blood, hemocyte lysate, lymph fluid and homogenate of lepidoptera silkworm moth insects as raw material liquid, and obtains the peptidoglycan recognition protein-D with electrophoretic purity and even HPLC purity by one or more than two of ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, salting-out or ultrafiltration methods;

or cloning the gene for encoding the peptidoglycan recognition protein-D into a recombinant expression vector, and introducing the recombinant expression vector into a host cell to obtain the recombinantly expressed peptidoglycan recognition protein-D.

Based on the above technical scheme, further, in the ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, salting-out or ultrafiltration method:

(1) Operating temperatures in the range from 0 ℃ to 45 ℃, preferably 0 ℃ to 10 ℃;

(2) The pH value of the solution is between pH2 and pH12, preferably between pH4 and pH10;

(3) The reagent for regulating the pH value of the solution is conventional and universal acid, alkali, acid solution or alkali solution, wherein the acid or acid solution is preferably HCl, HAc, phosphoric acid, citric acid, sulfuric acid, boric acid or a mixed solution thereof, and the alkali or alkali solution is preferably NaOH, KOH, tris, sodium citrate or potassium salt, sodium phosphate or potassium salt, borax or a mixed solution thereof;

(4) The buffer is a conventional and universal buffer ion pair buffer, preferably a citrate buffer ion pair, an HCl-Tris buffer ion pair, a citrate-phosphate buffer ion pair, a phosphate buffer ion pair, an acetate buffer ion pair, a borate-Tris buffer ion pair or a combination of the buffer ions;

(5) The ionic strength of the solution or buffer is in the range of 0.001mol/L to 0.5mol/L, preferably 0.01mol/L to 0.1mol/L.

In another aspect, the invention provides a method for preparing the derivative or analogue or active fragment of the peptidoglycan recognition protein-D, cloning a gene encoding the derivative or analogue or active fragment of the peptidoglycan recognition protein-D into a recombinant expression vector, introducing the recombinant expression vector into a host cell, and separating and purifying the recombinant expression vector to obtain the derivative or analogue or active fragment of the peptidoglycan recognition protein-D.

Based on the technical scheme, the expression system further comprises a prokaryotic system and an insect cell system, wherein host cells of the prokaryotic system are escherichia coli cells or bacillus subtilis cells; the host cells of the insect cell system are insect cells; the expression form is intracellular expression or secretion form expression.

In another aspect, the invention provides an antibody of said peptidoglycan recognition protein-D or a derivative or analogue or active fragment thereof, wherein said natural peptidoglycan recognition protein-D or said derivative or analogue or active fragment of peptidoglycan recognition protein-D is used as antigen to stimulate the immune system of mice or rats or rabbits or dogs or sheep or horses or cattle.

In another aspect, the invention provides the use of said peptidoglycan recognition protein-D or said peptidoglycan recognition protein-D derivative or analog or active fragment or said antibody for affecting pro-phenolate activation system, antimicrobial peptide synthesis, detection of microorganisms and their associated molecular patterns.

Based on the technical scheme, further, the related molecular modes comprise lipopolysaccharide, beta-1, 3-glucan, peptidoglycan, lipoteichoic acid and mannans.

Compared with the prior art, the invention has the following beneficial effects:

the natural and recombinant PGRP-D and part of the fragments thereof have the advantages of routine and simple obtaining method and high yield, and can be widely applied to the biomedical fields of prevention, detection diagnosis, treatment and the like aiming at microorganisms and microorganism related molecular modes.

Description of the drawings:

in order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described.

FIG. 1 shows the isolation and purification of natural PGRP-D, wherein Lane M: molecular weight markers; lane 1: natural PGRP-D purified by method 1; lane 2: method 2 purified natural PGRP-D; lane 3: method 3 purified natural PGRP-D.

FIG. 2 is an electropherogram of recombinant PGRP-D (prokaryotic expression system) isolated and purified, wherein Lane M: molecular weight markers; lane 1: PGRP-D fused with histidine tag after C terminal; lane 2: PGRP-D fused with histidine tag and thrombin cleavage site after C terminal; lane 3: PGRP-D fused with GST tag before N end; lane 4: PGRP-D with N-terminal pre-fused histidine-SUMO tag and thrombin cleavage site.

FIG. 3 shows an electropherogram of recombinant PGRP-D (insect expression system) separation and purification, wherein Lane M: molecular weight markers; lane 1: recombinant PGRP-D obtained by the pFastBac1-sf9 insect expression system; lane 2: PGRP-D obtained by pMIB/V5-His-Sf21 insect expression system.

FIG. 4 is an analysis of the binding capacity of PGRP-D to microorganisms, wherein (A): binding ability of native PGRP-D to different bacteria; (B): binding ability of recombinant PGRP-D to different bacteria.

FIG. 5 shows the immune correlation between PGRP-D mRNA expression level, wherein (A): changes in the time-varying expression of ApPGRP-D in vivo after bacterial induction; (B): changes in ApPGRP-D expression in various tissues after bacterial induction, mg in the figure: middle intestine; fb: fat bodies; mt: a mahalanobis tube; hc: blood cells; em: and (3) epidermis.

FIG. 6 is the effect of exogenous recombinant PGRP-D on the pro-phenoloxidase activation system, wherein HL: tussah hemolymph; PGRP: recombinant Ap-PGRP-D protein; MIC: a microorganism; error line is mean value + -standard deviation, experiment is repeated 3 times; * Represents t-test P <0.05,/represents t-test P <0.01,/represents t-test P <0.001,/represents t-test P <0.0001.

FIG. 7 shows the inhibition of the pro-phenoloxidase activation system by PGRP-D antibodies, wherein HL: tussah hemolymph; ab: rabbit Ap-PGRP-D polyclonal antibody; micro: a microorganism; error line is mean value + -standard deviation, experiment is repeated 3 times; * Represents t-test P <0.05,/represents t-test P <0.01,/represents t-test P <0.001,/represents t-test P <0.0001.

FIG. 8 is a graph of decreasing the inhibition of the pro-phenoloxidase activation system by endogenous PGRP-D, wherein DEPC: a control group injected with DEPC water; dsEGFP: injecting EGFP double-stranded RNA group; dsPGRP: injecting Ap-PGRP-D double stranded RNA; error line is mean value + -standard deviation, experiment is repeated 3 times; * Represents t-test P <0.01, t-test P <0.001, t-test P <0.0001.

FIG. 9 is a graph showing the effect of interfering with PGRP-D on the production of antimicrobial peptides, wherein (A): interference of PGRP-D on E.coli-induced antibacterial peptide production; (B): interference of PGRP-D on S.aureus induced production of antibacterial peptides; (C): the influence of PGRP-D on the generation of C.albicans induced antibacterial peptide is interfered, the error line is the mean value plus or minus standard deviation, and the experiment is repeated for 3 times; * Represents t-test P <0.05, ×represents t-test P <0.01.

The specific embodiment is as follows:

the following examples are intended to enable those skilled in the art to more fully understand the invention and are not intended to limit the scope of the claims in any way.

Example 1: isolation and purification of Natural PGRP-D

In this example, tussah is repeatedly washed with distilled water or deionized water, and haemolymph is collected at 10deg.C to-5deg.C by conventional methods such as wax disc method, centrifugation method, back blood vessel blood sampling method, perfusion method, squeezing, homogenizing method, reflection bleeding method, tearing method, cutting method, shearing method, and puncturing method.

1. Method 1

Haemolymph was collected, and the supernatant was collected after centrifugation at 12000 Xg. Performing 50% ammonium sulfate precipitation; centrifuging, taking the precipitate, and redissolving the precipitate by using 50mM citric acid buffer solution pH5.2, 100mM NaCl and 5% glycerol; centrifuging, collecting supernatant, passing through Sephacryl S-200 column, and collecting effluent component containing target protein; the fraction was dialyzed against 50mM PB pH8.0, and eluted with a 0-3M NaCl gradient through a PGRP antibody affinity column, and the effluent fraction containing the target protein was collected.

As a result of the test, the purity of the natural PGRP-D reached electrophoretic purity as shown in FIG. 1Lane 1.

2. Method 2

Dissolving in insect physiological saline (120 mM NaCl, 0.9mM CaCl) ₂ 、2.7mM KCl、0.5mM MgCl ₂ 、1.8mM NaHCO ₃ 、1mM NaH ₂ PO ₄ Fungus (Candida albicans), gram-positive bacteria (Micrococcus luteus) and gram-negative bacteria (Escherichia coli) mixture (10 μl) were injected into tussah body, induced for 24-48 hours, and then the fungus-induced haemolymph was collected, diluted 10-fold with 50mM Tris-HCl buffer pH5.5, passed through Mono-Q ion exchange chromatography column, and subjected to linear gradient elution with 0-3M NaCl in 50mM Tris-HCl buffer pH 5.5. The objective component was collected, concentrated to 1mL by ultrafiltration, diluted 10-fold with 10mM sodium phosphate buffer pH4.5, and subjected to linear elution by Octyl-sepharose4-Fast Flow and 10-500mM sodium sulfate buffer pH4.5 to obtain the objective protein component.

The test results are shown in FIG. 1Lane2, and the purity of the natural PGRP-D reaches electrophoretic purity.

3. Method 3

The Xylosmae haemolymph was precipitated with 70% ammonium sulfate, centrifuged at 8000 Xg for 15min and the supernatant was discarded. Re-dissolving the precipitate with phosphate buffer solution, loading onto hydroxyapatite column, gradient eluting with phosphate ion, and collecting the target protein component; dialyzing the above components with phosphate buffer solution, passing through anion exchange column HiTrapTM Q, eluting with NaCl concentration gradient (0-1M), and collecting target protein-containing component; addition of the above components (NH) ₄ ) ₂ SO ₄ To a concentration of 2M, a phenyl hydrophobic column was applied using (NH) ₄ ) ₂ SO ₄ Eluting with concentration gradient (0-50%) to collect target protein component; the components are subjected to PGN-sepharose affinity chromatography, and the target protein-containing component is collected.

As a result of the test, the purity of the natural PGRP-D reached electrophoretic purity as shown in FIG. 1Lane 3.

Example 2: PGRP-D structure analysis and gene sequence analysis thereof

According to the technology, method and means of conventional protein chemistry and molecular biology, PGRP-D is subjected to structural analysis to obtain the complete nucleotide sequence and amino acid sequence of PGRP-D.

The amino acid sequence of the natural PGRP-D (mature peptide chain) is shown in SEQ ID NO:1, the gene sequence of the coding natural PGRP-D is shown as SEQ ID NO: 2.

Obtaining the full-length cDNA sequence of PGRP-D by using molecular biology technology and method, and the sequence is shown as SEQ ID NO:3, the coded amino acid sequence is shown as SEQ ID NO: 4.

Example 3: recombinant PGRP-D and analogues and active fragments thereof obtained by using prokaryotic expression system

This example illustrates the strategy and basic methodology for the construction of prokaryotic expression systems for the expression of PGRP-D and its analogues, active fragment genes according to the invention.

PGRP-D derivatives, analogues, active fragment structures

(1) Met-PGRP-D amino acid sequence

MYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES

(2)Met-His ₆ tag-PGRP-D sequences

MHHHHHHYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES

(3)Met-PGRP-D-His ₆ Tag sequences

MYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAAC YTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSESHHHHHH

(4)Met-His ₆ Tag-thrombin cleavage site-PGRP-D sequence

MHHHHHHLVPRGSYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES

(5) Met-GST tag-thrombin cleavage site-PGRP-D sequence

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES

(6) Met-PGRP-D-thrombin cleavage site-GST tag sequence

MYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSESLVPRGSILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEGDEGDKWGNKKFELGLEFPNLPWYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD

(7) Met-PGRP-D-Flag tag sequence

MYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSESDYKDDDDK

(8) Met-Flag tag-MSPH sequence

MDYKDDDDKYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVI HHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES

(9)Met-His ₆ tag-SUMO tag-thrombin cleavage site-PGRP-D sequence

MHHHHHHSASGGTGDEDKKPNDQMVHINLKVKGQDGNEVFFRIKRSTQMRKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELEMEEGDEIDAMLHQTGGSCCTCFSNFLVPRGSYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSES

(10) Met-SUMO tag-thrombin cleavage site-PGRP-D-His ₆ Tag sequences

MHHHHHHSASGGTGDEDKKPNDQMVHINLKVKGQDGNEVFFRIKRSTQMRKLMNAYCDRQSVDMNSIAFLFDGRRLRAEQTPDELEMEEGDEIDAMLHQTGGSCCTCFSNFLVPRGSYPSIFSVESVGNEVPPYDFPFVSRSQWNARKPNETLPLQTPVPYVVIHHSATPAACYTKEECCIAMRSMQNFHIDGRRWWDIGYHFGVGSDATVYEGRGWSALGAHSLHFNSVSIGICVIGDWTGSLPPADQIKATKSLIAAGVDLGYIRPDYKLVGHRQVRATECPGDALYENIKTWPHYSAFPSSDKDLINVKELPESFRQKYFNKTKSESHHHHHH。

The expression vector, the expression host cell and the expression strategy of the prokaryotic expression system are conventional and universal expression vector, expression host cell and expression strategy of genetic engineering expression.

The method, principle, strategy, etc. of example 1 were used for the isolation and purification of the expression product.

Construction of PGRP-D expression vector

Respectively designing corresponding oligonucleotide primers according to the N-terminal and C-terminal amino acid sequences of the natural PGRP-D, and simultaneously respectively adding restriction endonuclease hydrolysis site sequences at the 5' ends of the two oligonucleotide primers; performing PCR amplification by using insect fat body cDNA pool as a template, detecting a product by agarose gel electrophoresis, and performing gel recovery of a nucleic acid fragment; carrying out restriction endonuclease digestion, carrying out double digestion on the plasmid, carrying out recombination connection under the action of DNA ligase, and thermally converting competent cells of the escherichia coli; the positive transformant is obtained through colony PCR and restriction endonuclease digestion, verification and screening, and then submitted to a biotechnology service company for DNA sequence determination. By the genetic engineering method, an expression vector of the PGRP-D gene is constructed.

The expression vector of this example was constructed as follows: 1. the escherichia coli is taken as a host, and the expression vectors can be selected from pTYB11, pMAL-C2X, pET-28a, pGEX-2T, pBV, pQE30, pET20b and the like; 2. a peptide fragment can be fused in front of the N end of PGRP-D to be used as a Tag (Tag) of affinity chromatography; 3. a peptide fragment can be fused after the C fragment of PGRP-D as a Tag (Tag) for affinity chromatography; 4. the Tag may be His-Tag (six or more histidines in succession), GST-Tag, flag-Tag, etc.; 5. amino acid sequences of proteolytic enzyme hydrolysis sites, such as thrombin, enterokinase, factor X, etc., may be added between the affinity chromatography tag and the PGRP-D to obtain recombinant PGRP-D protein with the same structure as the natural PGRP-D protein.

2. Recombinant PGRP-D protein and its derivative, analogue and active fragment obtaining

Transforming the PGRP-D gene expression vector into escherichia coli by utilizing a genetic engineering technology, picking single bacterial colonies, inoculating to LB containing antibiotics, and inducing the expression of the PGRP-D gene, thereby obtaining a culture solution or thalli containing the PGRP-D. The bacteria containing PGRP-D are firstly cracked by a lysate, broken by ultrasound, target proteins are released, and then the supernatant is collected by a centrifugal method to be used as a raw material liquid of recombinant PGRP-D for standby.

Characteristics of recombinant gene expression of interest: 1. the mode of transforming the expression vector into the host may be selected from a thermal transformation method and an electrotransformation method; 2. the mode of induction expression comprises chemical induction-isopropyl beta-D-thiogalactoside (IPTG) induction and heating induction; the PGRP-D gene may be expressed either intracellularly or extracellularly; 4. PGRP-D existing in cells needs to be released into the solution by means of lysis of lysate, ultrasonication and the like.

Recombinant PGRP-D and its derivatives, analogues, active fragments were isolated and purified to the desired purity from the above PGRP-D containing stock solutions according to the methods, principles, strategies, etc. of example 1 until electrophoretic purity or HPLC purity was achieved.

For example: (1) Constructing PGRP-D gene fused with histidine tag after C-terminal by adopting pET-19b, thermally transforming escherichia coli, and expressing PGRP-D-His in cells through IPTG induction. And (3) re-suspending the thalli by using a lysis buffer solution, performing ultrasonic disruption, and centrifuging to obtain a supernatant serving as a raw material liquid for further separation and purification of PGRP-D. PGRP-D was isolated and purified to electrophoretic purity (FIG. 2Lane 1) according to the method, principles, strategy, etc. of example 1.

(2) Constructing PGRP-D gene fused with histidine tag and thrombin after C terminal by pET-28a (+) and transferring the expression vector into host cells by an electrotransformation method, heating to induce expression, re-suspending thalli by using a lysis buffer (50 mmmol/LPBS, 0.15 mmmol/L NaCl,50 mmmol/L imidazole), performing ultrasonic crushing and centrifuging to obtain supernatant serving as raw material liquid for further separating and purifying PGRP-D. PGRP-D was isolated and purified to electrophoretic purity (FIG. 2Lane 2) according to the method, principle, strategy, etc. of example 1.

(3) Constructing a PGRP-D expression vector fused with a GST tag before the N end by adopting pGEX4T-1, thermally converting escherichia coli, and expressing the GST-PGRP-D in cells by heating induction. And (3) re-suspending the thalli by using a lysis buffer solution, performing ultrasonic disruption, and centrifuging to obtain a supernatant serving as a raw material liquid for further separation and purification of PGRP-D. PGRP-D was isolated and purified to electrophoretic purity (FIG. 2Lane 3) according to the method, principle, strategy, etc. of example 1.

(4) Constructing a PGRP-D expression vector with N-terminal pre-fused histidine-SUMO tag and thrombin cleavage site by adopting pET-28a-SUMO, thermally converting escherichia coli, and expressing the His-SUMO-thrombin cleavage site-PGRP-D in cells by heating induction. PGRP-D was isolated and purified to electrophoretic purity (FIG. 2Lane 4) according to the method, principles, strategy, etc. of example 1.

The purified expression product containing the tag is subjected to hydrolysis by the conventional and universal proteolytic enzyme (such as thrombin, enterokinase, coagulation X factor and the like), fusion peptide segments in the expression product are removed, and then the recombinant PGRP-D is obtained through separation and purification, wherein the structure of the recombinant PGRP-D is the same as that of the natural PGRP-D.

Example 4: recombinant PGRP-D and analogues and active fragments thereof obtained by using insect cell expression system

This example illustrates the strategy and basic methodology for constructing insect cell expression systems for expressing the PGRP-D and its analogs, active fragment genes of the invention.

The expression vector, the expression host cell and the expression strategy of the insect cell expression system are conventional and universal expression vectors, expression host cells and expression strategies of genetic engineering expression.

This example is intended to provide a more complete understanding of the invention to those skilled in the art, and is not intended to limit the scope of the invention in any way as set forth in the appended claims.

1. Recombinant PGRP-D and analogues and active fragments thereof obtained by using pFastBac1-sf9 insect expression system

PGRP-D and analogues thereof, and active fragment genes are connected into pFastBac1 plasmid to construct pFastBac1-PGRP-D recombinant expression plasmid. After induction of the transposable E.coli DH10, blu-gal and IPTG, the transposable recombinant bacmid was obtained by blue-white screening. Transfected insect cells sf9 and Western blot verifies that recombinant PGRP-D is expressed in cells.

The cells were collected, resuspended in lysis buffer (0.05 mol/L Tris-HCl,0.5mol/L NaCl, pH 8.0), sonicated and centrifuged to give a stock solution containing the target protein. Directly loading the recombinant protein onto an affinity chromatography column taking an anti-PGRP-D antibody-sepharose CL-6B as a ligand, and carrying out gradient elution by adopting a lysis buffer solution of 0mol/L-3mol/L NaCl to obtain the high-efficiency expression of the recombinant protein and achieve the electrophoretic purity. The result of the electrophoresis identification after purification is shown in FIG. 3-lane 1.

2. Recombinant PGRP-D and analogues and active fragments thereof obtained by using pMIB/V5-His-Sf21 insect expression system

PGRP-D and analogues thereof, and active fragment genes are connected into a pMIB/V5-His plasmid to construct a pMIB/V5-His-PGRP-D recombinant expression plasmid. After induction of the transposable E.coli DH5, blue-gal and IPTG, the transposable recombinant bacmid was obtained by Blue-white screening. Transfected insect cells Sf21 and Western blot verifies that recombinant PGRP-D is expressed in cells.

The cells were collected, resuspended in lysis buffer (0.05 mol/L Tris-HCl,0.5mol/L NaCl, pH 8.0), sonicated and centrifuged to give a stock solution containing the target protein. Directly loading the sample on a pre-balanced metal ion chelating chromatographic column, fully washing by 0.02mol/L imidazole (pH 8.0) to remove a large amount of impurity proteins, eluting by 0.2mol/L imidazole (pH 8.0), and obtaining high-efficiency expression of recombinant proteins to reach electrophoretic purity. The result of the electrophoresis identification after purification is shown in FIG. 3-lane 2.

Example 5: PGRP-D antibody acquisition

The various PGRP-D obtained in examples 1,3, 4 were used as antigens to stimulate the immune system of mice or rats or rabbits or dogs or sheep or horses or cattle to produce the corresponding antibodies according to conventional, general antibody production techniques.

The production of PGRP-D antibodies in the serum of immunized mice or rats or rabbits or dogs or sheep or horses or cattle is detected using conventional, universal antibody detection methods.

When the PGRP-D antibody is produced by the immunized mice or rats or rabbits or dogs or sheep or horses or cattle, the serum of the immunized mice or rats or rabbits or dogs or sheep or horses or cattle is collected and stored by adopting a conventional and universal animal serum collection and storage method, and the serum can be directly applied.

The PGRP-D antibodies with different purities are separated and purified from the stored serum containing the PGRP-D antibodies by using conventional and general antibody separation and purification technologies, such as salting out, various types of chromatographic media, antibody affinity chromatographic media and the like, until the PGRP-D antibodies with electrophoretic purity or HPLC purity are obtained, so that the method is suitable for different required applications.

Example 6: biological Activity of native PGRP-D and recombinant PGRP-D partial fragments

The same biological activity is achieved for the natural PGRP-D and recombinant PGRP-D active fragments in this example. The biological activity test insect of tussah as lepidoptera insect is described as representative. The application range of the active fragments of the natural PGRP-D and the recombinant PGRP-D can be further expanded by the skilled in the art by taking the biological activities of the active fragments of the natural PGRP-D and the recombinant PGRP-D as cores and bases.

1. Binding specificity of native PGRP-D and recombinant PGRP-D partial fragments to microorganisms

(1) Binding specificity of native PGRP-D to microorganisms

The binding properties of native PGRP-D with gram-positive bacteria (Staphylococcus aureus), gram-negative bacteria (Escherichia coli) and fungi (Saccharomyces cerevisiae) were examined using the western-blotting method. The method comprises the steps of respectively incubating natural PGRP-D with equal amount of microorganisms, eluting with 1M NaCl, eluting the components again with 2% SDS at high temperature (55 ℃), and indirectly detecting the combination condition of the natural PGRP-D and different types of microorganisms by using a PGRP-D polyclonal antibody. The results are shown in FIG. 4-A, where the natural PGRP-D binds to E.coli, staphylococcus aureus and Saccharomyces cerevisiae.

(2) Recombinant PGRP-D and its partial fragment and microorganism binding specificity

The binding properties of the recombinant PGRP-D partial fragments to gram-positive bacteria (Staphylococcus aureus), gram-negative bacteria (Escherichia coli) and fungi (Saccharomyces cerevisiae) were examined according to the western-blotting method described in example 6, 1- (1). As shown in FIG. 4-B, recombinant PGRP-D had binding to all three microorganisms, and the experimental results were consistent with those of the natural PGRP-D.

The above experiments demonstrate that native and recombinant PGRP-D is capable of specifically binding to gram-positive bacteria, part of gram-negative bacteria and part of fungi. The same experimental results were obtained with the recombinant PGRP-D partial fragments described in examples 3 and 4.

Correlation of PGRP-D expression level with innate immunity

After three representative microorganism equal proportion mixed solutions of E.coli, S.aureus and C.albicans are injected into a silkworm body, natural tussah tissues 18S rRNA are used as reference genes, and a Real-time PCR method is used for detecting the expression condition of PGRP-D in the tussah body at 3h, 6h, 9h, 12h, 18h and 24h, the results are shown in a figure 5-A, and the expression condition can be expressed in each tissue of the tussah, the results are shown in a figure 5-B, the expression quantity of PGRP-D mRNA in the tussah body gradually increases along with the change of the microorganism induction time, and the expression quantity reaches a peak value at 12 h; among the five tissues, PGRP-D mRNA was expressed in the epidermis most, and secondly, in the fat body, midgut and blood cells, was hardly expressed in the midgut. From the above experimental results, it can be seen that PGRP-D participates in the innate immune defense system of tussah and plays its role mainly in epidermis and fat body.

3. Influence of native, recombinant PGRP-D and antibodies thereto on the pro-phenoloxidase activation system

(1) Effect of recombinant PGRP-D and fragments thereof on the pro-phenoloxidase activation system

The effect of recombinant PGRP-D on the pro-phenoloxidase activation system was examined using six soluble PAMPs and three microorganisms, and the results are shown in FIG. 6, which shows that both PAMPs and microorganisms significantly activated PPO-AS compared to the control group of haemolymph+Tris-HCl buffer solution; after the exogenous recombinant PGRP-D is added, each experimental group shows the phenomenon of obviously increasing PO activity.

(2) Influence of in vivo interference of PGRP-D expression on the pro-phenoloxidase activation system

RNAi technology is adopted to reduce the expression of endogenous PGRP-D, and the influence of endogenous natural PGRP-D on PPO-AS is further examined. The results are shown in FIG. 8, where PO activity was significantly reduced in each experimental group following the natural PGRP-D interference, compared to the DEPC water injected control group and the dsEGFP control group.

(3) Effect of anti-PGRP-D antibodies on pro-phenoloxidase activation System

Endogenous native PGRP-D protein was blocked using rabbit anti-PGRP-D polyclonal antibodies and the effect of endogenous PGRP-D on the pro-phenoloxidase activation system was examined by six soluble PAMPs with three microorganisms. As shown in FIG. 10, PAMPs and microorganisms significantly activated PPO-AS compared with the control group of Xyloma tussah + Tris-HCl buffer; after the anti-PGRP-D polyclonal antibody is added, each experimental group shows the phenomenon of obviously reduced PO activity.

4. Effect of Natural and recombinant PGRP-D on the Synthesis of antibacterial peptides

In order to examine the influence of PGRP-D on the synthesis of the antibacterial peptide, the experiment is carried out by injecting E.coll and S.aureus into tussah larvae respectively after injecting dsPGRP-D for 72h to down regulate the expression of the endogenous PGRP-D, and examining the change condition of the mRNA level of the antibacterial peptide in the tussah larvae. The results are shown in FIG. 9, in which the mRNA level of the antimicrobial peptide Defensin, gloverin, attacin, lebocin was significantly reduced after injection of E.coli in the interference PGRP-D experimental group as compared with the control group. And the mRNA level of the antimicrobial peptide Defensin, gloverin, attacin, lebocin was significantly up-regulated after injection of s.aureus into the interfering PGRP-D experimental group. The above results indicate that PGRP-D has a significant difference in the effect on the synthetic pathways of antibacterial peptides induced by different pathogens.

Example 7: natural PGRP-D, recombinant PGRP-D active fragment and application of antibody thereof

This example is described as being representative of PGRP-D, and the active fragments of PGRP-D also have the same biological activity. Meanwhile, the biological activity test insect of tussah as lepidoptera insect is also described as representative. The application range of PGRP-D and recombinant PGRP-D active fragments and antibodies thereof can be further expanded by the skilled in the art by taking the biological activities of the PGRP-D and recombinant PGRP-D active fragments and antibodies thereof as cores and bases.

PGRP-D and its active fragments affect the pro-phenoloxidase activation system and the synthetic pathway of antibacterial peptides

PGRP-D and active fragments thereof are capable of activating the activation of pro-phenol oxidizing enzymes and have different effects on antimicrobial peptide synthesis induced by different microorganisms, as described in example 6. Based on this, it can be applied to the related fields of activation system using phenol oxidase and pro-phenol oxidation enzyme, and antibacterial peptide synthesis.

PGRP-D and its active fragments for detection of microorganisms

PGRP-D and its active fragments are capable of binding to a portion of the microorganism and its associated molecular patterns as described in example 5, based on which it is detected whether the sample contains the microorganism or its associated molecular patterns by detecting whether the microorganism binds to PGRP-D and its active fragments.

PGRP-D and application of active fragment antibody thereof

The antibodies against PGRP-D and the active fragments thereof obtained in example 5 were used for the immunodetection of PGRP-D in lepidopteran insect samples by conventional, general techniques, methods, etc., such as immunology and molecular biology. The method is also suitable for the immunodetection tracking analysis and the qualitative and quantitative detection analysis of samples in the process of preparing PGRP-D by separating and purifying lepidopteran insects. The experiments in this regard have been applied to the examples of the preparation of the above-described isolation and purification of active fragments of the natural, recombinant PGRP-D.

To any sample of microorganism to be detected, a sufficient dose of PGRP-D and its active fragment antibodies is added. According to the method for detecting microorganisms by PGRP-D and active fragments thereof in this example, the detection of microorganisms in a sample to be detected is performed. As a result, even if the amount of the microorganism detected in the sample is not detected (negative result), the experimental design was applied as a negative control group for detecting the microorganism in the sample.

As described in example 6, PGRP-D and its active fragments activate the activation of pro-phenol oxidase, while blocking PGRP-D with antibodies to PGRP-D and its active fragments significantly inhibits the activity of phenol oxidase. Based on this, the activity of phenol oxidase can be regulated by using PGRP-D and its active fragment antibody, and thus can be applied to the related fields using phenol oxidase and pro-phenol activation system.

The above results indicate that: the antibodies to PGRP-D and its active fragments, through binding to PGRP-D and its active fragments, shield the binding bioactivity to microorganisms and their related molecular patterns, thereby losing the original bioactivity of PGRP-D and its active fragments. The shielding principle based on the combination can be widely applied.

Claims

1. A peptidoglycan recognition protein-D having an amino acid sequence as set forth in SEQ ID NO: 1.

2. The peptidoglycan recognition protein-D of claim 1, wherein the peptidoglycan recognition protein-D is derived from Lepidoptera (Lepidoptera) insects of the family sarcinidae (samurn iidae) and is selected from one of tussah, castor, wild silkworm, tussah, amber, us tussah, ailanthus, wild silkworm, american wild silkworm, camphor, maple.

3. A gene encoding the peptidoglycan recognition protein-D of claim 1 or 2.

4. A peptidoglycan recognition protein-D gene according to claim 3, wherein the peptidoglycan recognition protein-D gene has a nucleotide sequence set forth in SEQ ID NO: 2.

5. The derivative or analogue or active fragment of peptidoglycan recognition protein-D according to claim 1 or 2, comprising an amino acid sequence as set forth in SEQ ID NO:1 and has the biological activity of peptidoglycan recognition protein-D.

6. The derivative or analogue or active fragment of peptidoglycan recognition protein-D according to claim 5, wherein the derivative or analogue or active fragment of peptidoglycan recognition protein-D is selected from the group consisting of Met-PGRP-D sequences, met-His ₆ tag-PGRP-D sequence, met-PGRP-D-His ₆ Tag sequence, met-His ₆ Tag-thrombin cleavage site-PGRP-D sequence, met-GST tag-thrombin cleavage site-PGRP-D sequence, met-PGRP-D-thrombin cleavage site-GST tag sequence, met-PGRP-D-Flag tag sequence, met-Flag tag-PGRP-D sequence, met-His ₆ tag-SUMO tag-thrombin cleavage site-PGRP-D sequence, met-SUMO tag-thrombin cleavage site-PGRP-D-His ₆ A tag sequence.

7. The method for producing peptidoglycan recognition protein-D according to claim 1 or 2, wherein the peptidoglycan recognition protein-D is obtained in an electrophoretic purity or even an HPLC purity by using one or a combination of two or more of hemolymph, blood, a hemocyte lysate, a lymph fluid, and a homogenate of an insect belonging to the family lepidoptera, as a raw material liquid, by one or a combination of two or more of ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, salting-out, and ultrafiltration;

8. The method for producing a derivative or analogue or active fragment of peptidoglycan recognition protein-D according to claim 5 or 6, wherein a gene encoding the derivative or analogue or active fragment of peptidoglycan recognition protein-D is cloned into a recombinant expression vector, introduced into a host cell, and isolated and purified to obtain the recombinantly expressed derivative or analogue or active fragment of peptidoglycan recognition protein-D.

9. The antibody of claim 1 or 2, or the derivative or analogue or active fragment of peptidoglycan recognition protein-D of claim 5 or 6, wherein the natural peptidoglycan recognition protein-D or the derivative or analogue or active fragment of peptidoglycan recognition protein-D is used as antigen to stimulate the immune system of mice or rats or rabbits or dogs or sheep or horses or cattle.

10. Use of peptidoglycan recognition protein-D according to claim 1 or 2 or a derivative or analogue or active fragment of peptidoglycan recognition protein-D according to claim 5 or 6 or an antibody according to claim 9 for influencing pro-phenoloxidase activation systems, antibacterial peptide synthesis, detection of microorganisms and their related molecular patterns.